hGH and bovine growth hormone ("bGH") are proteins of about 191 amino acids that are naturally synthesized in the anterior lobe of the pituitary. The molecular weight of the mature proteins is about 22,000 daltons, but they are initially made as pre-GHs with an extra 26 amino acids at the amino-terminus. This leader (or signal peptide) is normally cleaved during secretion by pituitary cells to release the mature form.
Several forms of mature bGH have been found in nature. The amino-terminus can vary (due to variation in the site of cleavage during secretion) so that the mature protein begins with either NH.sub.2 -Ala-Phe-Pro or NH.sub.2 -Phe-Pro, the latter referred to as "(des Ala) bGH". Additionally, the amino acid at bGH position 126 may be either Leu or Val, apparently as a result of allelic variation in the bovine population.
Exogenous administration of bGH to cattle increases milk production, feed efficiency, growth rate, and the lean-to-fat ratio, and decreases fattening time.
bGH has been produced by recombinant DNA techniques, see, e.g., Fraser, U.S. Pat. No. 4,443,539 (yeast); Buell, EP Appl. No. 103,395 (bacteria); Krivl, EP Appl. No. 193,515 (bacteria); Kopchick, EP Appl. No. 161,640 (encapsulated mouse cells implanted into animals); DeBoer, EP Appl. No. 75,444 (bacteria; gene modified to eliminate harmful secondary structure) and this has facilitated the production of analogues of bGH by site-specific mutagenesis. Thus, Aviv, GB No. 2,073,245 describes production of Met-Pro (des Ala) bGH, Met-Arg (des Ala) bGH, Met-Glu-Gly (des Ala) bGH, and des (Ala1-Phe2-Pro3-Ala4) bGH in E. coli. Brems et al., Proc. Natl. Acad. Sci. USA 85: 3367-71 (1988) reported preparation of the bGH mutant K112L, which extended the hydrophobic face of the third alpha helix of bGH. The bGH (96-133) fragment of this mutant was also prepared.
The biological activity of proteolytic fragments of bGH has also been studied. Brems et al., Biochemistry 26: 7774 (1987); Swislocki et al., Endocrinology 87: 900 (1970); Paladini et al., TIBS 256 (Nov. 1979). The fragment bGH (96-133) is superior in growth-promoting assays to bGH (1-95) and bGH (151-191). Hara et al., Biochemistry 17: 550 (1978); Sonenberg, U.S. Pat. Nos. 3,664,925 and 4,056,520; Chen and Sonenberg, J. Biol. Chem. 250: 2510-14 (1977). An octapeptide derived from the amino-terminus of bGH has been shown to have hypoglycemic activity, see Ng et al., Diabetes 23: 943-949 (1974), but it has no effect on growth. Similar results were observed with the fragment bGH (96-133). Graf et al., Eur. J. Biochem. 64: 333-340 (1976); Hara et al., Biochem. 17: 550-56 (1978).
Analogues of bGH have varied in growth-promoting activity, as have the known analogues of other GHs. However, a GH analogue having growth-inhibitory activity has not been previously been reported.
A variety of transgenic animals have been produced. Hammer et al., Nature 315: 680-638 (1985) (rabbits, sheep and pigs). Certain of these animals have been caused to express a GH, and increased growth of such transgenic animals has been reported. Palmiter et al., Nature 300: 611 (1982) microinjected the male pronucleus of fertilized mouse eggs with a DNA fragment containing the promoter of the mouse metallothionein I gene fused to the structural gene of rat GH. Several of the transgenic mice developed from the genetically modified zygote exhibited a growth rate substantially higher than that of control mice. (In effect, the genetically modified mouse serves as a test environment for determining the effect of the hormone on animal growth). Later, Palmiter et al., Science 222: 809 (1983) demonstrated that a similar enhancement of growth could be obtained in transgenic mice bearing an expressible hGH gene. A like effect is observed when hGH releasing factor is expressed in transgenic mice. Hammer, et a., Nature 315: 413 (1985).
hGH and bGH have also been expressed in transgenic animals. McGrane et al., J. Biol. Chem. 263: 11443-51 (1988); Kopchick et al., Brazil. J. Genetics 12: 37-54 (1989); Chen et al., J. Biol. Chem. 269: 15892-97 (1994). However, transgenic animals characterized by an exogenous gene which confers a reduced growth phenotype were hitherto unknown.
Abnormally high GH levels have been associated with a number of disorders. The two classic disorders which are directly caused by high levels of GH are acromegaly and gigantism.
Changes associated with acromegaly include coarsening of body hair, thickening and darkening of the skin, enlargement and overactivity of sebaceous and sweat glands such that patients frequently complain of excessive perspiration and offensive body odor, overgrowth of the mandible, cartilaginous proliferation of the larynx causing a deepening of the voice, and enlargement of the tongue. In addition, excess GH in these patients is responsible for proliferation of articular cartilage which may undergo necrosis and erosion and endoneural fibrous proliferation which causes peripheral neuropathies. Excess GH also increases tubular reabsorption of phosphate and leads to mild hyperphosphatemia. Many of these symptoms are also seen in patients with gigantism.
The hallmark of treatments for acromegaly and gigantism is their ability to lower insulin-like growth factor-1 ("IGF-1") in plasma and/or tissue through either destruction of the pituitary or drug treatment. The role of IGF-1 in GH-mediated disorders, such as acromegaly and gigantism is well recognized. Melmed et al., Amer. J. Med. 97: 468-473 (1994).
The mainstay treatment modalities for these two disorders are pituitary ablation, radiation treatment, and bromocriptine mesylate. Pituitary ablation is a surgical procedure and, like any surgical procedure, is associated with a significant risk of complications including mortality. There are also risks associated with radiation treatment of the pituitary as well. In addition, the efficacy of radiation treatment may be delayed for several years. Moreover, these treatment modalities are not specific against that part of the pituitary that produces GH and may adversely affect adjacent tissue as well. Bromocriptine mesylate is a dopamine like drug which suppresses the production of GH. Recently, octreotide, a long-acting somatostatin analog has also been used to treat patients with acromegaly and gigantism which is refractory to surgery, radiation, and/or bromocriptine mesylate. Somatostatin inhibits the release of GH releasing hormone from the hypothalamus. GH releasing hormone stimulates production of GH in the pituitary and its secretion.
Another disorder that has been associated with abnormal GH levels is diabetes mellitus (DM).
Characteristically, patients with poorly controlled DM have been found to have high levels of circulating GH. It has been shown that hypophysectomy could reduce diabetic hyperglycemia, thus strongly implicating the role of GH as an active component of the metabolic derangements of diabetes. Houssay and Biasotti, Rev. Soc. Argent. Biol. 6: 251-296 (1930). It has been suggested that hypersecretion of GH may be the cause as much as the consequence of poor diabetic control. Press et al., New England J. Med. 310: 810-814 (1984).
Most diabetics do not die of acute hyperglycemia. The overwhelming majority of diabetics die from complications associated with diabetes such as end organ failure. While diabetes affects almost all organs, heart and kidney failure are the most common causes of death. Other organs or systems that are commonly affected by DM are the eyes, the blood vessels and the nervous system. Patients with long standing diabetes will commonly have diabetic retinopathy, angiopathy and peripheral neuropathy. It is possible that normal GH secretion has a permissive role in patients predisposed to severe diabetic retinopathy. In such patients and in others in whom attempts to optimize glycemic control are unsuccessful, pharmacologic intervention may be beneficial not only in improving glycemic control but also in preventing severe proliferative diabetic retinopathy. Gerich et al., New England J. Med. 310: 848-850 (1984).
Proliferative diabetic retinopathy is one of the leading causes of blindness in the United States and ranks second only to senile macular degeneration as a cause of permanent blindness. Benson et al., Diabetic Retinopathy, Duane, T., (eds.), Harper & Row, Philadelphia, Pa., pp. 1-24. In juveniles with insulin dependent diabetes, there is no evidence of diabetic retinopathy up to 5 years. However, 27% of juveniles who have had diabetes for 5 to 10 years have diabetic retinopathy. Also 71% of juveniles who have had diabetes for longer than 10 years have diabetic retinopathy. Greater than 90% of juveniles who have diabetes for 30 years will ultimately have diabetic retinopathy. Also, the 5 year mortality rate for individuals blind from diabetic retinopathy is 36%, in which death generally is caused by cardiac or kidney complications.
The pathogenesis of proliferative diabetic retinopathy is believed to be mediated by GH. It has been shown that human GH stimulates proliferation of human retinal microvascular endothelial cells in the diabetic; proliferation of these cells is the primary cause of proliferative diabetic retinopathy. Rymaszewski et al., Proc. Natl. Acad. Sci. USA 88: 617-621 (1991). Thus, the involvement of GH in end organ damage in the diabetic is well established. Smith et al., Abstract of Presentation at ARVO meeting (May, 1995).
The kidneys are another organ that is affected by DM. Chen et al., Endocrinology 136: 660-667 (1995). One type of pathology seen in patients with diabetic nephropathy is glomerulosclerosis. Glomerulosclerosis is the sclerosis of mesangial cells which is preceded by mesangial cell proliferation. Glomerular cells are responsible for filtering the blood and thus directly affect kidney function.
Transgenic mice which express bGH have been shown to have enlarged glomeruli which progressed to a state of glomerulosclerosis. Thus, GH has been implicated in the development of diabetic glomerulosclerosis. Doi et al., Am. J. Pathol. 137: 541 (1990); Bell, Am. J. Med. Sci. 301: 195 (1991).
The hypothesis that high levels of GH are responsible for many of the proliferative types of diseases seen in diabetics is further supported by the fact that dwarfs with diabetes do not develop the proliferative types of diseases seen in normal-sized diabetics. Merimee et al., New England J. Med. 298: 1217-1222 (1978).